The invention relates generally to the field of cryobiology. More specifically, the invention relates to method of cryoprotection and novel cryoprotectant solutions and the principles that allow minimization of their toxicity, preferably without substantially weakening their ability to vitrify and to resist devitrification.
Cryopreservation refers to the preservation of systems containing living cells at temperatures below the normal freezing point of water (0xc2x0 C.). The history and literature of cryobiology, the field of science that attempts to understand low temperature biological phenomena and to improve methods of cryopreservation, is large and voluminous, and its successes have touched hundreds of millions of Americans and many others around the world in one way or another over the past 40 years or so. But despite its many impressive successes, cryobiology has still not found ways to cryopreserve transplantable kidneys, hearts, and livers (despite the high value such technology would have) or even many simpler systems, for unlimited times. In many cases, cells such as human and bull sperm and even human corneas can withstand freezing, but with undesirable amounts of injury. For example, up to 95% of human donor semen does not freeze well enough to be used clinically, bull sperm survival is substantially less than 100%, and frozen/thawed corneas perform sufficiently poorly that eye banks generally use simple cold storage in OptiSol(trademark) for short periods in preference to indefinite preservation that could greatly reduce costs and solve many logistic problems. Clearly, there are ample reasons to improve cryopreservation techniques, from both a humanitarian and a commercial viewpoint, yet this has not been accomplished to date.
Fahy proposed, in 1981-1984, that excellent cryopreservation of both simple and highly complex living systems could be attained without ice formation, a process termed vitrification, by using chemical agents known as cryoprotectants in extremely high concentrations (Fahy, Cryobiology 18: 617, 1981; Fahy and Hirsh, in: Organ Preservation, Basic and Applied Aspects, D. E. Pegg, I. A. Jacobsen, and N. A. Halasz, eds, MTP Press, Ltd., 1982, pp. 399404; Fahy et al, Cryobiology 21: 407-426, 1984). As Fahy further explained in 1986, xe2x80x9cAll of the postulated problems in cryobiology . . . can be solved in principle by the selection of a sufficiently high concentration of cryoprotectant . . . In the extreme case, all ice formation could be suppressed completely by using a concentration of cryoprotectant sufficient to ensure vitrification.xe2x80x9d Fahy, Cryobiology 23:1-13 (1986).
The potential market for tissue replacements of all kinds, once all problems of supply and rejection have been overcome, has been authoritatively estimated to be in the vicinity of $500 billion per annum. This potential can be realized through a combination of greatly improved control of rejection, enhanced retrieval of natural tissues and organs, and the development of artificial tissues and organs (either engineered tissues and organs or tissues and organs that are simply grown in the laboratory instead of in human bodies), but only if it also becomes possible to cryopreserve the vast number of tissues and organs required to meet this immense market.
Clearly, the value of minimum-toxicity solutions for cryopreservation is vast, and represents a problem that, until now, has not been solved despite the intensive efforts of cryobiologists around the world, who have worked extensively on cryopreservation since the report of the cryoprotective effects of glycerol in 1949 (Polge, Smith, and Parkes, Nature 164: 666, 1949). Fahy himself has described numerous efforts to improve his own vitrification solutions for tissue slices and organs, without progress since the adoption of the VS4-VS41A series around 1986 (Fahy, Levy, and Ali, Cryobiology 24: 196213, 1987; Fahy, Lilley, Linsdell, Douglas, and Meryman, Cryobiology 27: 247268, 1990; Fahy, Cryobiology 35: 344-345, 1997).
The present application provides cryoprotectant solutions having unprecedented non-toxicity even at higher total concentrations than have previously been contemplated, and while retaining good stability on warming and methods for designing additional examples thereof. By using the principle presented, the user has great flexibility in choosing variations for fine-tuning for the user""s particular needs. Further, the invention permits the design of entirely new cryoprotective substances in keeping with the new design principles disclosed herein. The inventors believe this new cryopreservation technology will allow successful preservation of most systems by freezing or by vitrification, especially since it consists not only of specific compositions but also involves several new general principles in cryobiology. The present invention also introduces novel cryoprotectants identified for specific uses based on the general principles and novel uses of previously known cryoprotectants. Both are valuable in identifying best-mode cryopreservation solutions.
The invention provides new theoretical and practical guidelines useful for creating minimum-toxicity vitrification solutions. The invention also provides specific families of vitrification solutions based on these new theoretical insights that have minimum toxicity and that are effective for dissimilar biological systems.
Another aspect of the present invention provides improved solutions for cryopreservation of cells, tissues, organs, artificial organs, artificial tissues, and non-living biological systems. The invention also provides specific families of freezing solutions based on the new vitrification solutions.
The invention provides cryoprotectant solutions and methods that also have applications in preservation by freezing point depression, supercooling, and cold storage. The invention also provides specific solutes and combinations of solutes that, when used in the proper way, have wholly unforeseen beneficial effects (reduced cryoprotectant toxicity, enhanced vitrification tendency, and enhanced resistance to devitrification).
New ways of, and new agents for, inhibiting the growth of ice crystals in aqueous solutions and in other contexts are described herein. The invention provides methods for scaling up the vitrification of small biological systems for use on large biological systems with minimal or no required increases in the concentrations of standard cryoprotectants. The invention further provides cryoprotectant solutions that, very surprisingly, have no gap between the concentration that vitrifies and the concentration that can be warmed at modest rates without devitrification.